In the name of God
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Nucleic acids extraction
By: Zahra Yazdani
Ph.D. student of medical immunology
Mazandaran university of medical science
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Types of nucleic acids
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What is Nucleic acids extraction?
Nucleic acids extraction is a process that used to purify DNA or RNA by using chemical or physical methods from a biological sample or separating nucleic acids from protein, cell membranes, and other cellular components.
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Brief history
Friedrich Miescher (1869)
isolation of the cell material “nuclei” or “nucleic acid” and developing a method for nucleic acid isolation
(RNA and DNA)
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Brief history
Stahl and Meselson (1956)
developing a full-function protocol for DNA extraction (The density gradient centrifugation protocol)
It was the first protocol described for isolating DNA from E.coli bacteria.
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Goal of nucleic acids extraction
It is vital to biology, especially biotechnology. It is the first step of different applications like fundamental research, disease diagnosis and therapeutic decision.
It is very important to define the unique characteristics of DNA like the shape, the size and function.
It is used in medical conditions investigation such as Down syndrome and cystic fibrosis. It is also helpful in case of identification if a person is a carrier of the disease or not. So, by DNA sequence in relation to diseases, It helped in finding out the molecular basis and cure for various diseases.
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Goal of nucleic acids extraction
In criminal investigations, nucleic acids extraction from samples (e.g., hair – skin – blood) is used to determine if a person is a suspect or not and also it can prove whether a person was in the vicinity of the crime scene.
It also used in Paternity Tests.
It can be useful in genetic engineering. For animals, DNA extraction is helpful for transforming and cloning animal’s DNA. For plants, DNA can be useful in identifying and extracting a specific gene in order to replicate in generations of plants.
It also helped in creating many vaccines (e.g., Hepatitis B vaccine), hormones (e.g., growth hormones and insulin), and enzymes.
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Stages of nucleic acids extraction
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Lysis of cell
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Lysis of cell
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Lysis of cell
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Lysis of cell
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Lysis of cell
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Removing the impurities and proteins from the sample
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Organic based methods
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PCI Method
separates nucleic acid
reduces the foaming
between interphase
digests proteins
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PCI Method
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Advantages and disadvantages
This method is considered one of the best methods for nucleic acids extraction.
The quantity and quality of DNA obtained by method are very good.
Unfortunately, this method is it is unsafe because Phenol is volatile and can cause burns. the chloroform can faint us. So, it requires training and to prepare and handle chemicals.
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CTAB method
The CTAB buffer removes polysaccharides and polyphenols effectively and gives excellent yield for plant DNA.
This method required extensive chemical preparation and additional techniques like tissue homogenization and the use of liquid nitrogen. It also takes a long time.
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Inorganic methods
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Step2 in organic and inorganic methods
Adding cold Isopropanol or Ethanol
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Adding Isopropanol or Ethanol
Notes:
If the DNA concentration in the sample is low, isopropanol may work better than ethanol to precipitate the available proteins. In addition, isopropanol is often used for precipitating DNA from large volumes as less alcohol is used.
The ethanol and isopropanol can also wash away the remaining salt residue. After being washed in alcohol and subjected to a centrifuge, the precipitated DNA protein will form a pellet, which can be washed in alcohol again, dried, and re-suspended in a Tris or TE buffer. Be careful not to over dry the sample, since this can denature the DNA; just leave the washed pellet on the lab table for a few minutes. If isopropanol has been used during the extraction instead of ethanol, the sample may not adhere as tightly to the tube and may require a longer drying time.
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Adding Isopropanol or Ethanol
Notes:
White precipitate is didn’t form
Low concentration DNA
Low concentration Isopropanol/Ethanol
Freezing DNA in -20
Using very cold Isopropanol/Ethanol
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Silica Gel Based methods
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Purification
Now that the DNA/RNA is separated from the aqueous component, it
can be cleaned even more thoroughly. This purification can be done by
rinsing the sample with more alcohol, which will clear any remaining
cellular debris. Once purified, the genomic DNA/RNA sample is typically
resuspended in HPLC grade water or an alkaline buffer so that it can be
easily transported, tested, or stored.
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What's the Best Way to Elute and Store Your DNA?
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TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer for preserving the stability of your preparation for a long time. Tris buffer controls the pH, while the EDTA chelates any divalent cations like Mg2+, preventing the activity of DNase. While this is very important to preserve the stability of your DNA, it will limit what you can do with these samples. This is because divalent cations are essential cofactors for many tools used in modern molecular biology like restriction enzymes and polymerases. In the situation where you need to use plasmid in TE for any enzymatic reaction, an effective possibility is to dilute your sample in water to reduce the EDTA concentration.
Alternatively, if the priority is to directly use your DNA preparation in any downstream application involving enzymatic reactions, a valid alternative can be Tris buffer (with no EDTA) or water. These are two of the choices that companies offer for the elution of plasmid DNA through their kit. The advantage of using a Tris buffer is that it allows better control of the pH preparation stabilizing the plasmid, and still allowing most downstream processing. On the other hand, nuclease free water is a great option because it provides a great versatility on how to use your plasmid in every type of experiment. The two solutions may differ though in their capacity to maintain DNA stability during storage over prolonged periods of time, especially at room temperature or at 4°C.
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Storing nucleic acids: temperature and longevity
Long time DNA:-20 °C or even -80°C for years
RNA: -80°C for 6 month
short period DNA: 4°C or even room temperature
RNA: -20°C
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Automatic Nucleic Acid Extraction System
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Safety and sterilization
RNase-Free – DEPC water
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Detection and Analysis of Nucleic Acids
UV Spectrometry
NanoDrop Spectrometry
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Determining Purity
The OD at 260nm/280 nm should be 1.6-2.00
If the 260nm/280nm ratio is <1.6 for DNA, <2.0-2.3 for RNA this indicates contamination, usually with protein.
DNA -If the OD ratio is higher than 2.0 it may be contaminated with RNA.
Ratio of the readings : O.D.260/O.D.280 is a measure of purity.
Pure preparations of DNA and RNA have O.D 260/280 of 1.8 and 2.0 respectively.
The A260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates)
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Detection and Analysis of Nucleic Acids
Fluorometric Methods
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Detection and Analysis of Nucleic Acids
Polymerase Chain Reaction (PCR) ?
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Any question?
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Thanks
for your
attention
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